LOCUS       sequence-476787-        5672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express SmBiT-VPS4A Fusion Protein in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5672)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5672)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: SmBiT-VPS4A Fusion Vector
FEATURES             Location/Qualifiers
     source          1..5672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        338..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          981..1113
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1096..1115
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1157..1176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1157..1175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2735..2856
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2746..2765)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2971..3004)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3014..3061
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3093..3887
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3147..3166)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3757..3776
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4045..4633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4125..4144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4759..4807
                     /note="synthetic polyadenylation signal"
     CDS             complement(4831..5229)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5226..5235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5260..5617)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5274..5409
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5426..5617
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5574..5594
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtt aattaagacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
       61 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
      121 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
      181 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
      241 tgacttcgta cgagagccta ggattatggc gcgccactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
      781 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctggttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt tatcacagtt
      901 aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc tgcagaagtt
      961 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat
     1021 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
     1081 ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct
     1141 cttaaggcta gagtattaat acgactcact atagggctag cgctcaccat ggtgaccggc
     1201 taccggctgt tcgaggagat tctcgggagc tccggtggtg gcgggagcgg aggtggaggc
     1261 tcgagcggtg cgatcgccat gacaacgtca accctccaga aagccattga tctggtgacg
     1321 aaagccacag aggaggacaa agccaagaac tacgaggagg cgctgcggct gtaccagcat
     1381 gcggtggagt acttcctcca cgctatcaag tatgaggccc acagcgacaa ggccaaggag
     1441 agcattcgag ccaagtgcgt gcagtaccta gaccgggccg agaagctgaa ggattattta
     1501 cgaagcaaag agaaacacgg caagaagcca gtcaaagaga accagagtga gggcaagggc
     1561 agtgacagtg acagtgaagg ggataatccg gagaaaaaga aactgcaaga acagctgatg
     1621 ggtgccgtcg tgatggagaa gcccaacata cggtggaacg acgtggccgg gctggagggg
     1681 gccaaggagg ccctcaaaga agctgtcatt ttgccaatca aattcccaca cttgttcaca
     1741 ggcaagcgca ccccctggcg ggggattctg ctgttcggac cccctggcac agggaaatcc
     1801 tacctggcca aagccgtggc aacagaggcc aacaactcca ccttcttctc tgtgtcctcc
     1861 tcagatctga tgtccaagtg gctgggggag agtgaaaagc tggtcaagaa cctgtttgag
     1921 ctggccaggc agcacaagcc ctccatcatc ttcatcgatg aggtggattc cctctgcggg
     1981 tcccgaaatg aaaatgagag tgaggccgcc cggaggatca aaacggagtt cttggtccag
     2041 atgcaggggg tggggaataa caatgatggg actctggttc ttggagccac aaacatccca
     2101 tgggtgttgg attcggccat caggaggagg tttgaaaaac gaatttatat ccccttgccg
     2161 gaggaagctg cccgcgccca gatgttccgg ttgcatctcg ggagcactcc ccacaacctc
     2221 acggatgcaa acatccacga gctggcccgg aagacggaag gctactcggg cgcggacatc
     2281 agcatcatcg tgcgggactc tctcatgcag cccgtgagga aggtgcagtc ggccacacac
     2341 ttcaaaaagg tctgtggccc ctctcgcacc aaccccagca tgatgattga tgacctcctg
     2401 actccatgct caccagggga cccaggagcc atggagatga cttggatgga tgtccctggg
     2461 gacaaactct tagagcctgt ggtttgcatg tcggacatgc tgcggtctct ggccaccacc
     2521 cggcccacgg tgaatgcaga cgacctcctg aaagtgaaga aattctcaga ggactttggg
     2581 caagagagtg tttaaacgaa ttcgggctcg gtacccgggg atcctctaga gtcgacctgc
     2641 aggcatgcaa gctgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     2701 caccgctgag caaggccgct tcgagcagac atgataagat acattgatga gtttggacaa
     2761 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     2821 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     2881 atgtttcagg ttcaggggga gatgtgggag gtttttttaa gcaagtaaaa cctctacaaa
     2941 tgtggtaaaa tcttaattaa gaattctaat ataacttcgt atagcataca ttatacgaag
     3001 ttatggatcc actgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     3061 caaggtgagg aactaaaccc aggaggcaga tcatgattga acaagatgga ttgcacgcag
     3121 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3181 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3241 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     3301 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3361 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3421 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3481 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3541 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3601 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3661 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3721 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3781 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3841 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgattc gaaatgaccg
     3901 accaagcgac gcccaaccgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3961 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4021 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4081 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4141 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4201 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4261 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4321 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4381 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4441 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4501 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4561 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4621 aaaaggattt caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4681 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4741 tttatagtcc ggaatcgtca cacaaaaaac caacacacag atgtaatgaa aataaagata
     4801 ttttattgcg gccatcgtga tggctagagt ttagccctcc cacacataac cagagggcag
     4861 caattcacga atcccaactg ccgtcggctg tccatcactg tccttcacta tggctttgat
     4921 cccaggatgc agatcgagaa gcacctgtcg gcaccgtccg caggggctca agatgcccct
     4981 gttctcattt ccgatcgcga cgatacaagt caggttgcca gctgccgcag cagcagcagt
     5041 gcccagcacc acgagttctg cacaaggtcc cccagtaaaa tgatatacat tgacaccagt
     5101 gaagatgcgg ccgtcgctag agagagctgc gctggcgacg ctgtagtctt cagagatggg
     5161 gatgctgttg attgtagccg ttgctctttc aatgagggtg gattcttctt gagacaaagg
     5221 cttggccatg gtggcgctag tgtcagaaga atcgagcttt ttgcaaaagc ctaggcctcc
     5281 aaaaaagcct cctcactact tctggaatag ctcagaggcc gaggcggcct cggcctctgc
     5341 ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag
     5401 gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct
     5461 ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt
     5521 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa
     5581 ctgacacaca ttccacagct ggttctttcc gcctcagaag gtacctaacc aagttcctct
     5641 ttcagaggtt atttcaggcc atggtgctgc gc
//
